Plasma membrane flipping of Syntaxin-2 regulates its inhibitory action on insulin granule exocytosis

Enhancing pancreatic β-cell secretion is a primary therapeutic target for type-2 diabetes (T2D). Syntaxin-2 (Stx2) has just been identified to be an inhibitory SNARE for insulin granule exocytosis, holding potential as a treatment for T2D, yet its molecular underpinnings remain unclear. We show that excessive Stx2 recruitment to raft-like granule docking sites at higher binding affinity than pro-fusion syntaxin-1A effectively competes for and inhibits fusogenic SNARE machineries. Depletion of Stx2 in human β-cells improves insulin secretion by enhancing trans-SNARE complex assembly and cis-SNARE disassembly. Using a genetically-encoded reporter, glucose stimulation is shown to induce Stx2 flipping across the plasma membrane, which relieves its suppression of cytoplasmic fusogenic SNARE complexes to promote insulin secretion. Targeting the flipping efficiency of Stx2 profoundly modulates secretion, which could restore the impaired insulin secretion in diabetes. Here, we show that Stx2 acts to assist this precise tuning of insulin secretion in β-cells, including in diabetes.


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The authors declare that all data supporting the findings of this study are available within the paper and/or the Supplementary Information/Source data file.
No sample size calculation was performed, and sample size was based on previous experience with single cells and human islets (PMID: 32051343, PMID: 28115395, PMID: 24835618, PMID: 28481223) as well as experience and common standards in similar field for calculating statistical significance and also dependent on donor availability.
Human pancreatic islets were purchased from the IsletCore, University of Alberta. Cadaveric donor pancreata deemed unsuitable for transplantation were obtained from Trillium Gift of Life Network, Canada. No selection criteria were applied.
Human pancreas obtained from normal portions of pancreatic operations. Human pancreatic islets were purchased from the IsletCore, University of Alberta, which were from institutional review board-approved donors with preoperative written informed consent for research by donors themselves and their family.
All animal procedures and use of human pancreas were carried out in accordance with ethical guidelines of the University of Toronto's Animal Care Committee and Research Ethics Board of the University Health Network, Toronto, ON, Canada, and with approved IRBs.
No sample-size calculation was performed. Sample sizes were decided based on previous publications (PMID: 28115395, PMID: 15339904, PMID: 32051343, PMID: 28031464, PMID: 32312960), experience and common standards in similar field for calculating statistical significance and also dependent on donor availability, a minimum of three independent experiments were carried out. Sample numbers are indicated in the figure legends. Multiple tests and analyses were performed as described in the manuscript to ensure the samples are representative and results are conclusive.
No data were excluded.
The number of replicates for each specific experiment is indicated throughout the manuscript text, figure legends and methods. All attempts of replication were successful.
Not relevant. For each group, islet samples from each donor were equally divided onto several coverslips that were treated differently only during data acquisition. All experiments were performed with appropriate negative and positive controls in keeping with the standards of the field.
Data files from individual donors were processed together and without knowledge of the treatment group (except for donor information, which was known on arrival of islet samples). !-actin, used as loading control, blocking buffer:10% Bovine Serum Albumin; Immunogen: Synthetic peptide. This information is proprietary to Abcam and/or its suppliers. Specificity Statement: The antibody reacts specifically against human, mouse and rat, which validated by western blots. This loading control is widely used by more than 3000 publications. Validation Data available on supplier website and our previous publication, PMID: 32051343, PMID: 28115395 as well as other's publication, .PMID: 33262832, 32963176.